Structure, function, and mechanism of human MIF and parasitic orthologs [Elektronische Ressource] / vorgelegt von Swen Zierow

Swen Klingelhöfer

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Structure, Function, and Mechanism
of Human MIF and Parasitic
Orthologs



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation


vorgelegt von

Diplom-Biologe
Swen Zierow

aus Furtwangen





Berichter: Universitätsprofessor Dr. Jürgen Bernhagen
Universitätsprofessor Dr. Klaus Wolf

Tag der mündlichen Prüfung: 19.12.08

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek
online verfügbar.



Table of Contents

STRUCTURE, FUNCTION, AND MECHANISM OF HUMAN
MIF AND PARASITIC ORTHOLOGS ....................................... 1
A. ABBREVIATIONS .................................................... I
B. ACKNOWLEDGMENTS ......... III
C. PUBLICATIONS ..................... V
1 INTRODUCTION ...................................... 6
1.1 PARASITES AND DISEASE ...................................... 6
1.1.1 Mechanisms of Immune Evasion ................. 7
1.2 LEISHMANIASIS .................................................... 8
1.2.1 Leishmania Life Cycle ................................ 10
1.2.2 Host Defense and Parasite Interactions .... 11
1.2.3 Treatment ................................................... 13
1.3 MALARIA ............................................................ 14
1.3.1 Plasmodium spp. Life Cycle ....................... 15
1.3.2 Host Defense and Parasite Interaction ..................................... 16
1.4 MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) ...................... 17
1.4.1 MIF is a Proinflammatory Cytokine .......... 17
1.4.2 MIF Receptors ............................................. 18
1.4.3 Apoptosis and Cell Proliferation ................................................ 19
1.4.4 Glucocorticoids ............ 20
1.4.5 MIF in Disease 21
1.4.6 The Three-Dimensional Structure of MIF ................................. 25
1.4.7 MIF, an Enzyme ......................................... 26
1.4.8 Small Molecule Inhibitors of MIF .............. 28
2 MATERIALS AND METHODS .............................................. 30
2.1 EQUIPMENT, CONSUMABLES AND CHEMICALS ................................... 30
2.1.1 Equipment .................................................. 30
2.1.2 Consumables ............... 31
2.1.3 Multi-Component Systems ......................................................... 31
2.1.4 Chemicals .................... 32
2.2 BACTERIA, YEAST, AND MAMMALIAN CELLS ...................................... 33
2.2.1 Bacteria ....................................................... 33
2.2.2 Yeast Cells .................. 33
2.2.3 Mammalian Cells ........................................ 34
2.3 PLASMIDS AND PRIMERS .................................... 34
2.3.1 Plasmids ...................................................................................... 34
2.3.2 Primers ........................ 34
2.4 MEDIA, BUFFER AND SOLUTIONS ....................... 35
2.4.1 Media for Bacterial Culture 35
2.4.2 Yeast Media ................................................................................ 35
2.4.3 Cell Culture Media ..... 36
2.4.4 Buffers and Solutions ................................................................. 36

2.5 CELL CULTURE TECHNIQUES ............................................................. 39
2.5.1 Isolation of PBMCs ..................................... 39
2.5.2 Chemotaxis Studies .... 39
2.5.3 Cellular Uptake Studies 39
2.6 METHODS IN MOLECULAR GENETICS/BIOLOGY .. 40
2.6.1 Transformation of Bacteria ........................................................ 40
2.6.2 Plasmid-DNA Extraction from Bacteria .................................... 40
2.6.3 Transformation of Yeast Cells (Yeast two Hybrid) ................... 40
2.6.4 Agarose Gel Electrophoresis ...................... 41
2.6.5 Isolation of DNA from Agarose Gels .......... 41
2.6.6 Digestion of DNA with Restriction Enzymes ............................ 41
2.6.7 Ligation of DNA fragments ........................................................ 42
2.6.8 Determination of DNA Concentration ....................................... 42
2.6.9 PCR ............................................................. 42
2.7 METHODS OF PROTEIN BIOCHEMISTRY .............. 43
2.7.1 Protein Expression ..................................... 43
2.7.2 Purification of Proteins .............................. 43
2.7.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........... 47
2.7.4 Coomassie Staining .................................... 47
2.7.5 Competition Binding Assay ........................................................ 47
2.7.6 BIAcore Analysis ........................................ 48
2.7.7 Determination of Protein Concentrations . 48
2.7.8 D-Dopachrome Tautomerization Assay ..................................... 49
2.7.9 Mass Spectrometry ..... 49
2.7.1 Steady-State Fluorescence Spectroscopy ... 50
2.7.2 NMR-Experiments ...................................... 50
2.8 METHODS OF STRUCTURAL BIOLOGY ................................................. 50
2.8.1 Protein Crystallization ............................... 50
2.8.2 Data Collection of LmMIF .......................... 51
2.8.3 Structure Determination of LmMIF .......... 51
2.8.4 Data Collection of Human MIF◦4-IPP Complex ........................ 52
2.8.5 Structure Determination of Human MIF Complexed with 4-
IPP ............................................................................................... 52
3 SPECIFIC AIM ........................................ 53
4 RESULTS ................. 54
4.1 STRUCTURE DETERMINATION OF HUMAN MIF IN COMPLEX WITH THE
NOVEL ACTIVE SITE INHIBITOR 4-IPP ............... 54
4.1.1 Crystallization of Human MIF Complexed with 4-IPP............. 54
4.1.2 The Three-Dimensional Crystal Structure of the MIF◦4-IPP-
Complex....................................................................................... 57
4.2 THE PARASITIC MIF ORTHOLOGS FROM LEISHMANIA MAJOR AND
PLASMODIUM FALCIPARUM ................................ 60
4.2.1 Sequence Alignment of LmMIF, PfMIF, and Human MIF ....... 60
4.3 PURIFICATION AND CHARACTERIZATION OF LMMIF .......................... 62
4.3.1 LmMIF Activity Studies ............................................................. 64
4.3.2 Crystallization of LmMIF ........................... 65

4.3.3 The Three-Dimensional Crystal Structure of LmMIF .............. 67
4.3.4 Human and Leishmania MIF Interaction Studies .................... 70
4.3.5 In vitro Binding of LmMIF to the Human MIF Receptor CD74
and to the Human MIF-tethering Protein p115 ........................ 73
4.3.6 Cellular Uptake of LmMIF ................................ 76
4.3.7 Chemotaxis Activity of LmMIF for Human Monocytes ............ 77
4.4 PURIFICATION AND CHARACTERIZATION OF PFMIF ........................... 78
4.4.1 In vitro Binding of PfMIF to the human MIF Receptor CD74 . 81
4.4.2 Crystallization of PfMIF............................................................. 81
4.5 MAPPING THE BINDING OF MIF TO THE CHEMOKINE RECEPTOR
CXCR4 .............................................................. 83
5 DISCUSSION ........................................... 86
5.1 THE PROTOTYPIC COVALENT MIF INHIBITOR 4-IPP .......................... 86
5.2 THE MIF ORTHOLOG FROM LEISHMANIA MAJOR ................................ 88
5.3 INTERACTION OF HUMAN AND LEISHMANIA MIF WITH THE
CHEMOKINE RECEPTOR CXCR4 ........................................................ 93
5.4 THE MIF ORTHOLOG FROM PLASMODIUM FALCIPARUM ..................... 96
6 SUMMARY ............................................... 98
7 REFERENCES ....................................... 100








A. Abbreviations
Abbreviations are also defined where they first appear in the text. For
amino acids the three letter code are used.

4-IPP 4-iodo-6-phenylpyrimidine
-10 Å Ångstrom (1 Å = 10 m)
ARDS Acute respiratory distress syndrome
CD74 Major histocompatibility complex, class II invariant
chain
CL Cutaneous leishmaniasis
D-dopachrome 2-carboxy-2,3-dihydroindole-5,6-quinone
DMEM Dulbecco’s modified Eagles’s medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
E. coli Escherichia coli
ELISA Enzyme-linked immunosorbant assay
ERK Extracellular signal-regulated kinases
FBS Fetal bovine serum
HPP Hydroxyphenylpyruvate
IFN Interferon
IL Interleukin
iNOS Inducible nitric oxide synthase
IPTG Isopropyl β-D-thiogalactopyranoside
ISO-1 (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole
acetic acid methyl ester
Jab1 Jun activation domain binding protein 1
K Dissociation constant d
kDa kilo-Dalton (1 kDa = 1.6605 ◦ 10-21 g)
LmMIF MIF ortholog from L. major
LPS Lipopolysaccharide
i

MAD Multiple Anomalous Dispersion
MAPK Mitogen activated protein kinase
MIF Macrophag